Integrins, as transmembrane heterodimeric receptors, have important functions in cell adhesion, migration, proliferation, survival apoptosis and signal transduction, in many physio- as well as pathophysiological settings. Characterisation of integrins and their ligand/antagonist binding is notoriously difficult, due to high integrin redundancy and ubiquity. Bypassing the intrinsic difficulties of cell-based integrin expression, purification and reconstitution, we present for the first time the synthesis of a heterodimeric integrin receptor and its assembly into a block-copolymeric membrane mimic. We present comprehensive data to demonstrate the synthesis of functionally active integrin αv β3, generated by in vitro membrane-assisted protein synthesis (iMAPS). This work represents the first step towards a robust and adaptable polymer-based platform for characterisation of integrin-ligand interactions.
Silica-based nanoparticles (SiNPs) are presented to harvest complex membrane proteins, which have been embedded into unilammelar polymersomes via in vitro membrane assisted protein synthesis (iMAP). Size-optimized SiNPs have been surface-modified with polymer-targeting antibodies, which are employed to harvest the protein-containing polymersomes. The polymersomes mimic the cellular membrane. They are chemically defined and preserve their structural-functional integrity as virtually any membrane protein species can be synthesized into such architecture via the ribosomal context of a cellular lysate. The SiNPs resemble 'heavy leg irons' catching the polymersomes in order to enable gravity-based generic purification and concentration of such proteopolymersomes from the crude mixture of cellular lysates.
Erythropoietin deficiency is an extensively researched cause of renal anemia. The etiology and consequences of shortened red blood cell (RBC) life span in chronic kidney disease (CKD) are less well understood. Traversing capillaries requires RBC geometry changes, a process enabled by adaptions of the cytoskeleton. These changes are mediated by transient activation of the mechanosensory Piezo1 channel, resulting in calcium influx. Importantly, prolonged Piezo1 activation shortens RBC life span, presumably through activation of calcium‐dependent intracellular pathways triggering RBC death. Two Piezo1‐activating small molecules, Jedi1 and Jedi2, share remarkable structural similarities with 3‐carboxy‐4‐methyl‐5‐propyl‐2‐furanpropanoic acid (CMPF), a uremic retention solute cleared by the healthy kidney. We hypothesize that in CKD the accumulation of CMPF leads to prolonged activation of Piezo1 (similar in effect to Jedi1 and Jedi2), thus reducing RBC life span. This hypothesis can be tested through bench experiments and, ultimately, by studying the effect of CMPF removal on renal anemia.
Semiconductor
quantum dots (QDs) are widely used for optical applications
and bioimaging. In comparison to organic dyes used for fluorescent
labeling, QDs exhibit very high photostability and can be further
surface modified. Equipping QDs with magnetic properties (mQDs) makes
it possible to combine fluorescence and magnetic resonance imaging
analyses. For this purpose, we have prepared water-dispersible and
magnetic CdTe/ZnS mQDs, whereby ferrous ions are selectively incorporated
in either their cores or their shells. This study aims at understanding
the differences in optical, structural, and magnetic properties between
these core- and shell-doped mQDs. Field-dependent isothermal magnetic
susceptibility measurements show that shell-doped mQDs exhibit paramagnetic
and their core-doped equivalents superparamagnetic behavior
near room temperature. Shell doping results in about 1.7 times higher
photoluminescence quantum yields and 1.4 times higher doping efficiency
than core doping. X-ray diffraction patterns reveal that core doping
leads to defects in the lattice and hence to a severe decrease in
crystallinity, whereas shell doping has no significant impact on the
crystal structure and consequently fewer disadvantages regarding the
mQD’s quantum yield. These selective doping approaches, particularly
shell doping, allow for the tailored design of paramagnetic QDs having
modifiable and biocompatible particle surfaces. The organic ligandsin
this study N-acetyl-l-cysteinesufficiently
prevent leakage of toxic metal ions, as shown by cytotoxicity assays
with HepG2 cells. Confocal laser scanning microscopy shows that mQDs
are internalized by these cells and accumulated near their nuclei.
This study shows that biocompatible, fluorescent, and paramagnetic
QDs are promising photostable labels for multimodal bioimaging.
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